Preparation of sensors on oligo- or poly (ethylene glycol) films on silicon surfaces

ABSTRACT

A sensor that includes a) a silicon (Si) substrate having a surface; and b) a monolayer of oligoethylene glycol (OEG) bonded to the surface via silicon-carbon bonds. Regions of the OEG monolayer distal to the surface are functionalized with a molecular probe serving as a recognition element for a bioanalyte. A method of making a silicon surface that recognizes a biological specimen includes 1) hydrosilylating with a mixture that includes an oligoethylene glycol (OEG) substituted with an alkene at one end of the OEG and capped at the opposing end of the OEG and an oligoethylene glycol (OEG) substituted with an alkene at one end of the OEG and an alkyne having a protecting group at the opposing end of the OEG and 2) removing the protecting group from the alkyne; and 3) reacting the alkyne with a reagent in a 1,3-dipolar cycloaddition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/108,217, filed Apr. 23, 2008, which is a continuation-in-part of U.S.patent application Ser. No. 11/587,232 filed Oct. 23, 2006 which is thenational stage entry of PCT No. PCT/US05/14391 filed Apr. 27, 2005,which in turn claims priority to U.S. Provisional Application No.60/566,120 filed Apr. 18, 2004. This application also claims priority toU.S. Provisional Application No. 60/913,431, filed Apr. 23, 2007. Allthe aforementioned applications are incorporated by reference herein intheir entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.CTS-0210840, awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND

Microarray technology has been widely used for genomics and proteomicsresearch as well as for drug screening. Currently, the spot size in mostmicroarrays is larger than one micron. The use of nanometricbiomolecular arrays, with smaller spot sizes, will enablehigh-throughput screening of biomolecules—eventually at the singlemolecule level. Also, nanometric arrays permitting precise control overthe position and orientation of individual molecules will become apowerful tool for studying multi-valent and/or multi-component molecularinteractions in biological systems. Toward these ends, protein arrayswith feature sizes smaller than 100 nm have been fabricated, mostlyusing dip-pen nanolithography and nanografting. [See Lee et al., Science2002, vol. 295, p. 1702; Wilson et al., Proc. Natl. Acad. Sci. USA 2001,vol. 98, p. 13660; Liu et al., Proc. Natl. Acad. Sci. USA 2002, vol. 99,p. 5165; Pavlovic et al., Nano Lett. 2003, vol. 3, p. 779; and Krämer etal., Chem. Rev. 2003, vol. 103, p. 4367.]

Modification of silicon surfaces with organic thin films to allow strongand highly specific interactions with targeted biological entities is oftremendous interest in the fields of biomicroelectrical mechanicalsystems (bioMEMS) that may integrate biosensing with controlled deliveryof drugs. Target molecules interacting with the film surfaces can bedetected optically, mechanically, magnetically, electronically or thecombination. [Grayson, A. C. R.; Shawgo, R. S.; Johnson, A. M.; Flynn,N. T.; Li, Y. W.; Cima, M. J.; Langer, R. “A BioMEMS review: MEMStechnology for physiologically integrated devices.” Proc. IEEE 2004, 92,6-21.]

BioMEMS are of tremendous interest for their potential applications inmicroscale, high throughput biosensing and medical devices [Shawgo etal., J. Curr. Opin. Solid State Mater. Sci. 2002, v. 6, p. 329]. Usingsilicon as a substrate for the preparation of such devices isparticularly attractive, since the extensive micro-fabricationtechniques developed by the microelectronic industries can be used tofabricate and integrate various micro-components into the devices. Forreducing biofouling, considerable research has been directed to themodification of substrate surfaces with stable and ultrathin films ofpoly(ethylene glycol) (PEG) or oligo(ethylene glycol) (OEG) [Prime etal., Science 1991, vol. 252, p. 1164]. Since many of the ultimateapplications for bio-devices require moderate-term (e.g., a few hours toseveral days) exposure to biological media (e.g., buffer of pH 7.4 at37° C.), stability of the bio-compatible coatings on the devices underthese conditions is highly desirable. All of the OEG/PEG-terminatedfilms on silicon substrates reported by others are bound onto thesilicon surfaces via Si—O bonds that are prone to hydrolysis[Calistri-Yeh et al., Langmuir 1996, v. 12, p. 2747), thereby limitingtheir stability under physiological conditions (Sharma et al., Langmuir2004, v. 20, p. 348].

For implantable bioMEMS, inflammatory responses often lead to devicefailure due to the formation of a thick layer of fibrous cells on theimplant. [Wilson, G. S.; Gifford, R. “Biosensors for real-time in vivomeasurements.” Biosens. Bioelectron. 2005, 20, 2388-2403.] The initialstep of inflammatory response is the non-specific adsorption of proteinsonto the substrates. This step alone may greatly lower the sensitivityand specificity of the implanted sensor. Therefore, ideal coating forsilicon based biosensors should be 1) ultrathin for high sensitivity; 2)resistant to non-specific interactions with proteins and cells in bodyfluids and tissues; 3) strongly and specifically interacting with targetmolecules or cells; 4) stable over a period of time required by specificapplications under in vivo conditions.

In addition to bioMEMS applications, ultraflat, stable and highlyprotein-resistant films on silicon surfaces represent ideal platformsfor fabrication of single molecule arrays presenting signaling andadhesion molecules. These well-defined model systems allow forfundamental study of cell response to chemical signals at a singlemolecule level. A deeper understanding of how the nanoscale presentationof such molecules determine cellular functions, such as differentiation,proliferation and apoptosis, [Arnold, M.; Cavalcanti-Adam, E. A.; Glass,R.; Blummel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P.“Activation of integrin function by nanopatterned adhesive interfaces.”ChemPhysChem 2004, 5, 383-388; Maheshwari, G.; Brown, G.; Lauffenburger,D. A.; Wells, A.; Griffith, L. G. “Cell adhesion and motility depend onnanoscale RGD clustering.” J. Cell Sci. 2000, 113, 1677-1686.] is oftremendous importance for designing the next generation biomaterials,implantable bioMEMEs and pharmaceuticals.

SUMMARY

The present disclosure is generally directed to sensors, and to methodsof making such sensors.

In some aspects, embodiments disclosed herein relate to a sensor thatincludes a) a silicon (Si) substrate having a surface; and b) amonolayer of oligoethylene glycol (OEG) bonded to the surface viasilicon-carbon bonds. Regions of the OEG monolayer distal to the surfaceare functionalized with a molecular probe serving as a recognitionelement for a bioanalyte. The molecular probe is covalently bonded inthese regions as a cycloadduct of a 1,3-dipolar cycloaddition reaction.In other aspects, embodiments herein related to compositions having useas catalysts for a 1,3-dipolar cycloaddition reaction. A compositionincludes Cu(I) and a triazole ligand having an oligoethylene glycolsubstitutent.

A method of making sensors, in accordance with embodiments disclosedherein, involves 1,3 dipolar cycloaddition reactions between a molecularproble possessing a functional group (or precursor) 1,3 dipolar speciesand a carbon-carbon unsaturation displayed at the distal end of anoligoethylene glycol attached at the proximal end to a silicon surface.The present disclosure is also directed to methods of using such sensorsin arrays, especially in biosensors.

In other aspects, embodiments disclosed herein relate to a method ofmaking a silicon surface that recognizes a biological specimen. Themethod includes 1) hydrosilylating with a mixture that includes anoligoethylene glycol (OEG) substituted with an alkene at one end of theOEG and capped at the opposing end of the OEG and an oligoethyleneglycol (OEG) substituted with an alkene at one end of the OEG and analkyne having a protecting group at the opposing end of the OEG and 2)removing the protecting group from the alkyne; and reacting the alkynewith a reagent in a 1,3-dipolar cycloaddition. The reagent in the1,3-dipolar cycloaddition includes a portion capable of being recognizedby a biological specimen.

The foregoing has outlined rather broadly the features of the presentinvention in order that the detailed description of the invention thatfollows may be better understood. Additional features and advantages ofthe invention will be described hereinafter which form the subject ofthe claims of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, and theadvantages thereof, reference is now made to the following descriptionstaken in conjunction with the accompanying drawings, in which:

FIG. 1 depicts, schematically, a method of making nanometricbiomolecular arrays, in accordance with embodiments disclosed herein;

FIG. 2 depicts height (2 a, 0.5×0.5 μm², 10 nm contrast) and friction (2b, 0.1 V contrast) AFM images of an EG₇ film on Si (111) after AFManonization lithography, and a 3-dimensional image (2 c, 5×5 μm²) of apatterned area upon treatment with succinic anhydride, DMAP andpyridine;

FIG. 3 depicts AFM height (3 a, 3 c, and 3 e) and friction (3 b and 3 d,corresponding to 3 a and 3 c) images (4×4 μm²) of an area similar tothat shown in FIG. 2 c upon sequential treatment with EDAC/avidin (3a-b), biotinylated-BSA (3 c-d), and avidin (3 e), wherein the lines in 3a-b are used as guides;

FIG. 4 demonstrates, schematically, a nanofabrication process, inaccordance with embodiments of the present invention;

FIG. 5 illustrates a setup for AFM anodization lithography, inaccordance with embodiments of the present invention;

FIG. 6 illustrates AFM anodization lithography of an OEG monolayer on aSi surface, wherein the patterned (anodized) regions comprise functionalmoieties such as carboxylic acid, aldehyde, and/or alcohol, etc.,

FIG. 7 shows a scheme for preparing OEG-presenting monolayers on siliconby surface hydrosilylation using the OEG-terminated alkenes;

FIGS. 8 a-b show fluorescence image of an OEG (a) and amannose-presenting monolayer (b) on silicon upon incubation with E. coli(strain 83972) followed by fixing with formaldehyde and staining withpropidium iodide;

FIG. 9 shows a scheme for derivitizing C≡C-TMG groups;

FIGS. 10 a-b shows tapping mode AFM images of the TMG-protected alkyneon Si (111) surface: (A) 7.0×7.0 μm; (B) 4.1×4.1 μm;

FIGS. 11 a-d show survey and high resolution spectra of theTMG-protected alkyne on Si (111): (A) Survey spectrum; (B) Highresolution C1 s scan; (C) High resolution Ge 3d scan; (D) Highresolution Si 2p scan;

FIG. 12 shows N 1 s, F 1 s and Ge 3d region of XPS profile of filmsderived from 5 before (circle) and after (solid) click reaction with 6;

FIG. 13 shows survey and high resolution spectra before and after thesurface click reaction on Si(111), wherein the inset shows highresolution scans on the F1s and N1s regions before and after thereaction;

FIGS. 14 a-b show high resolution scans of the functionalized Si (111)surface after the click reaction: (A) Si 2p (B) N 1;

FIGS. 15 a-b show tapping mode AFM images of the resulting film afterclick reaction of the alkyne terminated Si(111) surface and the fluorideEG-6 functionalized azide: (A) 3.2×3.2 μm; (B) 3.5×3.5 μm;

FIG. 16 shows The effect of varying concentration of copper catalyst onthe percent conversion of the click reaction performed on Si (111);

FIG. 17 shows high resolution spectra of Cu 2p of clicked surfaces on Si(111) with and without EDTA.

FIG. 18 shows comparison of the percent conversion of the clickedmaterials on Si (111) surface with and without the addition of a ligand(Top);

FIG. 19 shows an optimized scheme;

FIGS. 20 a-b show fluorescence images after incubation of filmspresenting mannose in E. coli Hu2545 (a) and Hu2634 (b);

FIG. 21 shows a tris(triazole) 1 and an OEG-modified ligand 2;

FIG. 22 shows a scheme for synthesis of the water soluble Cu(I) ligand 2the Alkyne 10 and the Azides 14, 18, and 21;

FIG. 23 shows a scheme for CuCCA reactions of the Alkyne 10 with theAzides 14, 18, and 21;

FIG. 24 shows ligands Screened for CuAAC; and

FIG. 25 shows a scheme for synthesis of the Azide 15 for Screen theLigands of CuAAC reactions.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with embodiments of the present disclosure, a sensorincludes a) a silicon (Si) substrate having a surface; and b) amonolayer of oligoethylene glycol (OEG) bonded to the surface viasilicon-carbon bonds. Regions of the monolayer distal to the surface arefunctionalized with a ligand serving as a recognition element for abioanalyte. This ligand is generally covalently bonded to the OEGterminal end distal to the silicon surface as a cycloadduct of a1,3-dipolar cycloaddition reaction.

In some embodiments, the ligand is a biomolecule such as carbohydrates,proteins, oligonucleotides, and combinations thereof. In a particularembodiment the biomolecule is mannose. Utilizing mannose as the ligand,the sensor is capable of recognizing E. Coli as the bioanalyte.

In some embodiments, a method of modifying a silicon surface includeshydrosilylating with a mixture of an oligoethylene glycol (OEG)substituted with an alkene at one end of the OEG and protected at theopposing end of the OEG and an OEG substituted with an alkene at one endof the OEG and an alkyne having a protecting group at the opposing endof the OEG. This hydrosilylation bonds the OEG to the silicon surfacevia a carbon silicon bond. After removing the protecting group from thealkyne a 1,3-dipolar cycloaddition is performed.

The 1,3-dipolar cycloaddition may utilize an azide, a nitrile oxide, anazomethine ylide, a carbonyl ylide, or a nitrone. In particularembodiments, the 1,3-dipolar cycloaddition is executed with an azide.1,3-dipolar species that can participate in these reactions arecharacterized as three atom zwitterionic compounds having a resonancestabilized form that distributes a formal positive charge at one end(atom 1 or 3) of the three atom array and a formal negative charge atthe opposite end (atom 3 or 1) of the three atom array.

In still further embodiments, the present disclosure provides a methodof bioconjugation to a silicon surface that includes 1) hydrosilylatingwith a mixture that includes an oligoethylene glycol (OEG) substitutedwith an alkene at one end of the OEG and protected at the opposing endof the OEG and an oligoethylene glycol (OEG) substituted with an alkeneat one end of the OEG and an alkyne having a protecting group at theopposing end of the OEG 2) removing the protecting group from thealkyne; and 3) reacting the alkyne with a reagent in a 1,3-dipolarcycloaddition. The reagent in the 1,3-dipolar cycloaddition may includea modified biomolecule. The modified biomolecule may include a reactivefunctional group such as an azide, a nitrile oxide, an azomethine ylide,a carbonyl ylide, or a nitrone. In particular embodiments the modifiedbiomolecule has an azide as the reactive functional group.

The methods described herein may be useful in making a silicon surfacethat recognizes a biological specimen. To do so the method mayinclude 1) hydrosilylating with a mixture comprising: an oligoethyleneglycol (OEG) substituted with an alkene at one end of the OEG and cappedat the opposing end of the OEG; and an oligoethylene glycol (OEG)substituted with an alkene at one end of the OEG and an alkyne having aprotecting group at the opposing end of the OEG; 2) removing theprotecting group from the alkyne; and 3) reacting the alkyne with areagent in a 1,3-dipolar cycloaddition. The reagent in the 1,3-dipolarcycloaddition includes a portion capable of being recognized by abiological specimen.

The 1,3-dipolar cycloaddition further includes the use of a reactivefunctional group, for example, an azide, a nitrile oxide, an azomethineylide, a carbonyl ylide, or a nitrone. In particular embodiments, thereactive functional group is an azide. The biological specimen may be abacterium, a virus, a protein, a DNA sequence, a RNA sequence, or anoligosaccharide.

A silicon surface modified to interact with a biological specimen madeby the process comprising: hydrosilylating with a mixture comprising: anoligoethylene glycol (OEG) substituted with an alkene at one end of theOEG and capped at the opposing end of the OEG; and an oligoethyleneglycol (OEG) substituted with an alkene at one end of the OEG and analkyne having a protecting group at the opposing end of the OEG;removing the protecting group from the alkyne; and reacting the alkynewith a reagent in a 1,3-dipolar cycloaddition; wherein the reagent inthe 1,3-dipolar cycloaddition comprises a portion capable of beingrecognized by a biological specimen.

In some embodiments, methods of generated an array of biomolecules on aSi surface includes: (a) contacting OEG-terminated alkenes with ahydrogen-terminated Si surface to form a contacted surface; (b)photolyzing the contacted surface to effect Si—C bonding between theOEG-terminated alkenes and the Si surface and form an OEG-coated Sisurface comprising a monolayer of OEG bound to the Si surface throughSi—C bonds; (c) anodizing regions on the top of the OEG monolayer of theOEG-coated Si surface via AFM anodization lithography to yield ananolithographically-patterned OEG-coated Si surface comprising regionswith enhanced associability toward biomolecules; and (d) depositing atleast one type of biomolecule in the regions of enhanced associabilityto form a nanometric biomolecular array.

Generally, the nanometric biomolecular arrays made by theabove-described methods comprise: (a) a Si substrate; (b) a monolayer ofOEG bonded to the Si substrate via Si—C bonds, wherein regions at thetop of the monolayer have been lithographically-patterned; and (c)biomolecules associated with the lithographically-patterned regions ofthe OEG monolayer.

In some embodiments, the present invention provides a novel approach forpreparation of nanometric protein arrays, based on binding ofbiomolecules to nano-templates generated by AFM anodization lithographyon robust, ultrathin monolayers terminated with oligo(ethylene glycol)(OEG) derivatives with the general formula of —(CH₂CH₂O)_(n)—R (n>1,R═CH₃, H, etc.) on conducting silicon surfaces. A specific example isthe preparation of nanometric avidin arrays. Applicants have shown thatbiotinated-BSA, but not the native BSA, binds to the avidin arrays, andthe resulting arrays of biotinated-BSA can bind avidin to form proteindots with a feature sizes of ˜30 nm, scalable down to the size of asingle protein molecule.

Such nanometric arrays have at least the following unique advantages:(a) they will vastly improve the detection sensitivity (down to a singlemolecule), allowing for detection of biomolecular variations correlatedwith diseases, which are typically expressed at very low level; (b) theywill tremendously increase the probe density on a chip (e.g.,incorporating the whole human genome in the same chip); (c) they permita label-free detection of the binding of target molecules on thenanoarrays; (d) they greatly shorten the time for binding of targetmolecules to the nanoarrays and improve the efficiency of the binding;(e) they may substantially improve the specificity of the detection; (f)the single molecule arrays will greatly facilitate single moleculesequencing of DNA using polymerase and nucleotides that arefluorescently labeled, and single molecule arrays of the template andthe polymerase will reduce the background fluorescence and greatlyimprove the quality of the data; and such (g) nanometric arrays willbecome a powerful research tool for studying the cooperative interactionamong multiple biomolecules. It should be noted that advantages (a)-(e)can be gained only for nanoarrays where the spot size is less than about25 nm and comprising only one or a handful of probe molecules.

In some embodiments, the present disclosure is generally directed tonanometric biomolecular arrays and to a novel approaches for preparationof such nanoarrays, based on binding of biomolecules, such as avidin, totemplates generated by AFM anodization lithography (conductive AFM) onbiocompatible ultrathin films on silicon substrates. In someembodiments, such films are generally robust, ultrathin monolayersterminated with oligo(ethylene glycol) (OEG) with the general formula of—(CH₂CH₂O)_(n)—R (n>1, R═CH₃, H, etc.) on conducting silicon surfaces,wherein such films have been nanolithographically-patterned usingconductive AFM lithography (Maoz et al., Adv. Mater. 2000, vol. 12, p.725). The lithography process is followed by chemical and biochemicalderivatization of the resulting nanopatterns. The unique features ofthis approach include: (a) the OEG-monolayers resist non-specificadsorption and denaturing of proteins on the templates; (b) conductiveAFM can be used to selectively oxidize the top portion of theOEG-monolayer to generate carboxylic acids, aldehydes, alcohols andother functional groups that can be used to attach biomolecules; (c) themonolayers are attached to silicon substrates via Si—C bonds with a highdensity, rendering the system highly robust; and (d) the lithographyprocess is very rapid. In a specific example, the resulting avidinarrays have a feature size of ˜26 nm, and they can serve as templatesfor the preparation of nanoarrays of a wide variety of proteins that aresite-specifically labeled with biotin [Lue et al., J. Am. Chem. Soc.2004, v. 126, p. 1055].

As described in commonly assigned, co-pending U.S. patent applicationSer. No. 10/742,047, olig(ethylene glycol) (OEG) terminated alkenes weregrafted onto hydrogen-terminated silicon surfaces throughhydrosilylation (as developed by Linford and Chidsey, see Linford etal., J. Am. Chem. Soc. 1993, v. 115, p. 12631; Buriak, Chem. Rev. 2002,v. 102, p. 1271) forming robust Si—C bonds with the silicon surfaces. Itwas shown that the alkyl monolayers grown by this method were stable inboiling organic solvents, water, and acids, as well as slightly basicsolutions [Linford et al., J. Am. Chem. Soc. 1995, v. 117, p. 3145]. Amethod describing the modification of hydrogen-terminated siliconsurfaces, including a silicon atomic force microscopy (AFM) cantilevertip, with OEG-terminated alkenes via either thermally- or photo-inducedhydrosilylation is also found in commonly assigned, co-pending U.S.patent application Ser. No. 10/742,047. [See also Yam et al., J. Am.Chem. Soc. 2003, v. 125, p. 7498; Yam et al., Chem. Commun., 2004, p.2510]. The efficiency with which such OEG-terminated films resistprotein adsorption depends on many factors including the number ofethylene glycol (EG) units and the packing density of the films that isdetermined by the underlying substrate surface and the depositionmethods. For example, OEG-terminated thiolate self-assembled monolayers(SAMs) on gold (111) surfaces are protein resistant, but those on silver(111) surfaces are not [Herrwerth et al., J. Am. Chem. Soc. 2003, vol.125, p. 9359]. The latter was attributed to the high packing density andstructural ordering of the SAMs. Research has demonstrated that filmsgrown on Si(111) surfaces had a density similar to that of thecorresponding thiolate SAMs on gold (111) surfaces, and similarlyreduced the adsorption of fibrinogen to 1% monolayer or less.

Referring to FIG. 1, in some embodiments, such above-mentioned methodsgenerally comprise the steps of: (Step 101) contacting OEG-terminatedalkenes with a hydrogen-terminated Si surface to form a contactedsurface; (Step 102) photolyzing the contacted surface to effect Si—Cbonding between the OEG-terminated alkenes and the Si surface and form aOEG-coated Si surface comprising a monolayer of OEG bound to the Sisurface through Si—C bonds; (Step 103) lithographically anodizingregions on the top of the OEG monolayer of the OEG-coated Si surface viaAFM anodization lithography to yield a nanolithographically-patternedOEG-coated Si surface comprising regions with enhanced associabilitytoward biomolecules; and (Step 104) depositing at least one type ofbiomolecule in the regions of enhanced associability to form ananometric biomolecular array.

In some embodiments, the OEG-terminated alkenes comprise EG sequencesselected from the group consisting of EG₁-EG₂₀, and combinationsthereof, wherein “n” in EG_(n) describes the number of —(CH₂CH₂O)—repeat units. In some or other embodiments, the OEG-terminated alkenescomprise PEG-terminated alkenes, wherein PEG-terminated alkenes compriseEG_(n) sequences of n>20.

Typically, the Si surface is atomically flat. In some embodiments the Sisurface is selected from the group consisting of (100), (111), andcombinations thereof. In some embodiments, upon coating the Si surfacewith an OEG monolayer, the OEG-coated Si surface is washed, andoptionally dried, prior to lithographically anodizing regions on top ofit.

In some embodiments, the nanolithographically-patterned (anodized)regions of the OEG-coated Si surface comprise nanowells (i.e., “spots”).In some embodiments, the nanolithographically-patterned regions of theOEG-coated Si surface comprise carboxylic acid, aldehyde, alcohol,and/or other moieties, wherein these moieties provide, at least in part,the enhanced associability toward biomolecules.

In some embodiments, the at least one type of biomolecule is selectedfrom the group consisting of proteins, oligonucleotides, andcombinations thereof. Avidin is an exemplary such biomolecule. In someembodiments, at least some of the at least one type of biomolecule bindswith the regions of enhanced associability via amide bonds.

Generally, the nanometric biomolecular arrays made by theabove-described methods comprise: (a) a Si substrate; (b) a monolayer ofOEG bonded to the Si substrate via Si—C bonds, wherein regions at thetop of the monolayer have been lithographically-patterned; and (c)biomolecules associated with the lithographically-patterned regions ofthe OEG monolayer.

Typically, the above-mentioned Si surface is atomically flat. In someembodiments, the Si surface is selected from the group consisting of(100), (111), and combinations thereof. In some embodiments, the OEGbound to the Si surface comprises EG sequences selected from the groupconsisting of EG₁-EG₂₀, and combinations thereof. As mentioned above,such OEG is bound to the surface through Si—C bonds.

In some embodiments, the biomolecules (as part of the array) areselected from the group consisting of proteins, oligonucleotides, andcombinations thereof. Avidin is an exemplary such molecule. Typically,the biomolecules are associated by a bonding means selected from thegroup consisting of covalent bonding, ionic bonding, electrostaticforces, and combinations thereof. In some particular embodiments, thebiomolecules are associated with the lithographically-patterned regionsof the OEG monolayer via amide bonds.

In some embodiments, the nanometric biomolecular array is operable forbinding biomolecular analyte, i.e., it can be used to assay biomolecularanalyte, wherein biomolecular analyte can comprise one or more of avariety of different biomolecules. In such embodiments, biomolecularanalyte is deposited on the array, and the array is analyzed todetermine the regions in which the biomolecular analyte exhibits abinding affinity. In some such embodiments, the biomolecules andbiomolecular analyte are removed by treatment with proteinase K, whereinthe proteinase K serves to catalyze hydrolytic fragmentation of proteinsbound to the patterned OEG monolayer surface to regenerate the pattern.Biomolecular analyte suitable for such analysis (including sequencing)include, but are not limited to, oligonucleotides, proteins, andcombinations thereof. In some or other embodiments, such an array isuseful for screening drug candidates.

The monolayers described herein can be readily prepared from-hepta-(ethylene glycol)methyl ω-undecenyl ether, comprising seven EG(EG₇) repeat units (the term “EG₇” being used herein to describe boththe repeat units and the alkene precursor comprising the seven EG repeatunits), and conductive silicon (111) substrates with an atomically-flat,H-terminated surface (Yam et al., Chem. Commun., 2004, p. 2510). Inaccordance with some embodiments, AFM anodization lithography on thesemonolayers was performed under ambient conditions with a relativehumidity of ˜20-80%, using a Nanoscope IIIa AFM (Digital Instrument)equipped with a pulse generator. During AFM anodization on eachlocation, a short pulse of +(4 to 17) V, with a duration typically inthe range of about 10 nanoseconds (ns) to about 10 microseconds, wasapplied to the sample while the tip was grounded. During AFManodization, the AFM scanner can rapidly position the AFM tip on thesample with nanometer precision. This process for generating ahigh-density nanoarray proved to be much faster than dip-pen ornanografting lithography techniques that normally take seconds togenerate each nanospot. The pulse generator was then disconnected, andheight and friction AFM images were simultaneously acquired in contactmode at a 90° scan angle with the same tip. In one specific example, inwhich the nanolithography was performed with 100 ns pulses of +10 V, itwas found that holes with an apparent depth of 0.4 nm (corresponding tothe length of one ethylene glycol unit) were generated, as revealed bythe height images of the patterned areas, e.g., FIG. 2 a. However, thecorresponding friction image (FIG. 2 b) shows the presence of spots of˜10 nm in diameter where the friction is higher than the surroundings,indicating the presence of polar head groups on the spots. Referring toFIG. 2, the spacing between the spots was ˜50 nm, as controlled by thescanner.

While not intending to be bound by theory, it has been suggested thatAFM anodization of alkyl siloxane monolayers on silicon under certainconditions could oxidize the head groups of the monolayers intocarboxylic (COOH) groups [Maoz et al., J. Adv. Mater. 2000, v. 12, p.725]. However, a recent study of a similar system using secondary ionmass spectroscopy showed no signs of COOH groups on the oxidizedsurfaces [Pignataro et al., Mater. Sci. Eng. C. 2003, v. 23, p. 7].

It should be noted that the monolayers in the above-described study hadalkyl head groups, while the monolayers of the present inventioncomprise an OEG head group. The AFM anodization of the OEG-coatedsurfaces could be substantially different from the above-described alkylsurfaces. Preliminary studies by Applicants indicate that, upon AFManodization of OEG-coated surfaces, a variety of species includingcarboxylic acids, aldehydes, and alcohols, are generated on the filmsurface. Additionally, in some embodiments, treatment of an AFM-anodizedmonolayer with avidin is done in the presence of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC, which serves tomediate the formation of amide bonds between the surface COOH groups andthe protein molecules). Corresponding AFM images obtained during thefirst few scans showed that the protein molecules predominately adsorbedon the patterned spots. The protein molecules were readily removed bythe scanning tip afterwards indicating that the protein molecules werenot covalently bound to the surface. Again, while not intending to bebound by theory, it was concluded that rather than COOH groups, thesurfaces of the oxidized spots mainly comprised hydroxyl groups thatcould be chemically converted into COOH groups, e.g., by treatment withsuccinic anhydride, dimethyl-aminopyridine (DMAP) and pyridine.Patterned spots were “etched” upon this treatment forming nanoholes asshown by the three-dimensional AFM height image (FIG. 2 c). As measuredby the line profile of about 100 patterned spots in FIG. 2 c, thediameter of the holes was 91±6 nm, and the depth was 1.31±0.12 nm, aboutone third of the thickness of the film. Using the method describedherein, COOH groups were generated in the nanoholes, which may be usedto attach proteins. Upon incubation of the samples with EDAC followed byavidin in PBS solution, the nanoholes were nearly filled (FIG. 3 a) andbarely recognized even by comparison with the corresponding frictionimage (FIG. 3 b). The depth of the holes decreased to 0.43±0.06 nm,while the width of the holes remained nearly the same (87±9 nm).

The sample was then treated with BSA in PBS buffer. The depth of theholes remained the same, indicating that BSA did not bind to themolecules in the holes. To verify that the molecules in the holes wereindeed avidin, the sample was treated with a solution of biotinated-BSAin PBS buffer. AFM height and friction images (FIGS. 3 c and 3 d) revealthat the patterned spots protrude slightly from the film surface. Theheight and half-height width of the spots were 0.14±0.14 nm and 24±3.5nm, respectively. The fact that the molecules in the holes boundbiotinated-BSA but not native BSA is a strong indication that thesemolecules were avidin.

The patterned biotinated-BSA, with an average of nine biotin groups oneach BSA molecule, should have free biotin groups available for bindingadditional avidin onto the pattern. Indeed, upon incubation of thesample in a solution of avidin in PBS, nano-dots arrays were formed, asshown by the AFM height image (FIG. 3 e). The heights of the dots were1.27±0.37 nm and the half-height widths of the dots were 26±3.4 nm.While the top avidin molecules could be removed by repeated scanning,the protein molecules in the holes were strongly bound, and could not beremoved by the scanning tip, neither by immersion in PBS for 6 hours norin detergent (SDS) solutions for 14 hours. AFM images of the proteinarrays remained nearly the same after four weeks under ambientconditions. Upon treatment with Proteinase K (to catalyze the hydrolyticfragmentation of the proteins), nanoholes very similar to those in FIG.2 c were regenerated. The nanofabrication process, in accordance withsome embodiments of the present invention, is demonstrated in FIG. 4.

Nanometric biomolecular array fabrication, as described herein and inaccordance with embodiments of the present invention, will vastlyimprove the detection sensitivity (down to a single molecule), greatlyfacilitate single molecule sequencing of DNA, and serve as a powerfulresearch tool for studying the cooperative interaction among multiplebiomolecules.

The following examples are provided to demonstrate particularembodiments of the present invention. It should be appreciated by thoseof skill in the art that the methods disclosed in the examples whichfollow merely represent exemplary embodiments of the present invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments described and still obtain a like or similar result withoutdeparting from the spirit and scope of the present invention.

EXAMPLE 1

This example serves to illustrate materials used in the fabrication ofnanometric protein arrays, on protein-resistant monolayers on siliconsurfaces, in accordance with embodiments of the present invention.

Pyridine, succinic anhydride, 4-dimethylaminopyridine (DMAP),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), avidin, bovineserum albumin (BSA), biotinamidocaproyl labeled BSA (biotin-BSA),proteinase K, and phosphate buffered saline (PBS buffer, 0.01 Mphosphate, 0.14 M NaCl, pH 7.4) were purchased and were used withoutpurification.

EXAMPLE 2

This example serves to illustrate the synthesis of hepta(ethyleneglycol)methyl co-undecenyl ether (comprising EG₇), as used in someembodiments of the present invention.

Monomethyl hepta(ethylene glycol) (1.637 g, 4.81 mmol) was slowly addedto NaH (0.81 g, 33.75 mmol) in dry THF (8 ml) while stirring under N₂.To this mixture was added Bu₄NI (0.81 g, 0.48 mmol) and11-bromo-1-undecene (4.2 ml, 16.78 mmol), and the mixture was refluxedfor 20 hours under N₂. Iodomethane (3.42 g, 24.1 mmol) was added, andthe mixture was refluxed for 1 hour. The reaction mixture was thenrefluxed with methanol for another hour. After cooling to roomtemperature, the mixture was concentrated under reduced pressure.Dichloromethane was added, and the mixture was subsequently poured intowater. The organic layer was separated, and the aqueous layer wasextracted twice with dichloromethane. The combined organic layers werewashed twice with water, dried with magnesium sulfate, filtered andconcentrated under reduced pressure. The crude product was purified byflash chromatography (ethyl acetate/hexane/methanol 50:48:2) to affordEG₇ (1.8 g, 76%). ¹H NMR (CDCl₃): δ=5.80-5.82 (m, 1H); 4.90-5.01 (m,2H); 3.52-3.65 (m, 26H); 3.41-3.46 (t, 2H); 3.37 (s, 3H); 2.02-2.04 (q,2H); 1.54-1.57 (m, 4H); 1.30-1.36 (m, 12H). ¹³C NMR (CDCl₃): δ=139.35,114.22, 72.06, 71.67, 70.70, 70.65, 70.18, 59.17, 33.93, 29.76, 29.67,29.60, 29.55, 29.25, 29.05, 26.21. ESI-MS: 516.5 (100%, M+1+Na⁺).

EXAMPLE 3

This example serves to illustrate photo-induced surface hydrosilylation,in accordance with embodiments of the present invention.

H-terminated silicon (100 or 111) surfaces were prepared usingprocedures similar to those described by Hines and Chidsey [Krämer etal., Chem. Rev. 2003, vol. 103, p. 4367]. Briefly, single-sided polishedsilicon (100) or silicon (111) wafers with a resistivity less than 5ohm·cm were cut into pieces of ca. 1×1 cm², cleaned with NH₄OH/H₂O₂/H₂O(v/v 1:1:4) at 80° C. for 20 minutes, thoroughly washed withMillipore-purified water, etched in 10% buffer-HF for 10 minutes andthen in 40% NH₄F for 10 minutes under N₂ purge, and dried with a flow ofnitrogen. The setup and procedures for photo-induced surfacehydrosilylation of H-terminated silicon substrate surfaces with alkeneswere described in detail elsewhere [Yam et al., Chem. Commun., 2004, p.2510]. Briefly, a freshly prepared H—Si (100) or H—Si (111) substratewas placed inside a freshly cleaned and dried quartz cell, and tiltedwith the polished surface facing a droplet (˜1 mg) of the alkene (EG₇)that was placed on a surface in the cell. After the cell was degassed at˜0.1 mTorr for 10 minutes, the wafer was allowed to fall down onto thedroplet, sandwiching a thin and homogeneous layer of the alkene betweenthe substrate and the quartz wall. The substrate was then illuminatedwith a hand-held 254 nm UV lamp (Model UVLS-28, UVP) for 30 minutes,followed by washing sequentially with petroleum ether, ethanol, andCH₂Cl₂, and finally drying with a stream of N₂.

EXAMPLE 4

This example serves to illustrate how Si surface type can affect thestability of the resulting nanometric biomolecular array.

Resistance comparisons between the adsorption and stability ofOEG-terminated thin films on H—Si (100) and Si (111) were performedusing the method as described in the present invention. Resultsindicated that the films of -hepta-(ethylene glycol)methyl co-undecenylether, EG₇, on Si (111) and (100) substrates reduced adsorption ofprotein (fibrinogen) by >99%. The films were stable under a wide rangeof conditions, such as in biological buffers at pH 7.4 and 9.0 (37° C.),water (100° C.), and 2.5 M H₂SO₄ (100° C.). The films derived on Si(111) were more stable than those on Si (100). Furthermore, it wasdemonstrated that the films on Si (100) or Si (111) could be patternedby AFM anodization lithography by the method as described in the presentinvention. The resultant patterns may serve as templates for directingthe self-assembly of biomolecules such as fibrinogen, avidin, and bovineserum albumin (BSA) on the surfaces. See Yam et al., J. ColloidInterface Sci., 2005, in press.

EXAMPLE 5

This example serves to illustrate AFM anodization lithography onEG₇-coated Si (100) or Si (111) substrates, in accordance withembodiments of the present invention.

A setup for performing AFM anodization lithography on EG₇-coated Si(100) or Si (111) substrates, in accordance with embodiments of thepresent invention, is illustrated in FIG. 5. An OEG-coated silicon (100)or silicon (111) wafer was mounted on a steel sample puck using adouble-sided carbon conductive tape that was also used to attach ashort, thin Pt wire (25 μm in diameter). Another short, thin Pt wire wasconnected to the metal clip of a tip holder. The use of thin wires forelectrical connection to the wafer and tip greatly reduces the vibrationintroduced to the system. Both Pt wires were connected through a BNCcable to a digital delay/pulse generator equipped with a homemadeamplifier. The steel sample puck was mounted onto the AFM scanner thatwas insulated with a thin parafilm. AFM anodization lithography wasperformed under ambient conditions with a relative humidity of 25-55%,using a silicon cantilever with a force constant of 0.3 N/m andresistivity of ˜0.08 ohm·cm. During scanning of the sample in contactmode (load: ˜1 nN; scan size: 5×5 μm; scan rate: 29.8 μm/s), bursts of10 pulses of +17 V square waves (pulse duration: 1 μs; interval betweentwo pulses: 8.33 ms; interval between two bursts: 4.29 seconds) wereapplied to the sample while the tip was grounded. The nanolithographywas completed in one scan, taking 85 seconds. For relocation of thenanopatterned areas after subsequent ex situ treatment of the sample, atiny X mark with a line width of ˜8 μm was drawn on the substrate with adiamond pen before nanolithography, and the position of the cantileverrelative to the mark shown by the CCD camera of the AFM was recorded.FIG. 6 illustrates the above-described AFM anodization lithography of anOEG monolayer on a Si surface, wherein the patterned (anodized) regionscomprise carboxylic acid moieties.

EXAMPLE 6

This example serves to illustrate AFM imaging, in accordance withembodiments of the present invention.

After nanolithography, the pulse generator was disconnected, andtopography and friction AFM images were simultaneously acquired incontact mode at 90° scan angle with the same tip used fornanolithography. For imaging protein-coated surfaces, a soft cantileverwith a force constant of 0.03 N/m was used at a loading force of ˜1 nN.

EXAMPLE 7

This example serves to illustrate derivatization of the nanoarrays withbiomolecules, in accordance with embodiments of the present invention.

The patterned substrates were treated sequentially with (a) a solutionof succinic anhydride (100 mg), and DMAP (12 mg) in pyridine (1 ml) for30 minutes; (b) a solution of EDAC (1 mg) and avidin (0.1 mg) in PBS (1ml) for 5 minutes; (c) BSA (1 mg) in PBS (1 ml) for 5 minutes; (d)biotin-BSA (1 mg) in PBS (1 ml) for 5 minutes; (e) avidin (0.1 mg) inPBS (1 ml) for 5 minutes; and (f) proteinase K (1 mg) in PBS (1 ml) for3 hours. All treatments were carried out under ambient conditions. Aftereach step, the substrates were thoroughly washed with Millipore water,dried with a stream of N₂, and immediately imaged by AFM.

FURTHER EXAMPLES

Biocompatibility and Stability Study of OEG Films on Silicon

Protein-resistant thin films have been a subject of extensive research.Among many types of protein-resistant thin films, those presenting poly-or oligo(ethylene glycol) (PEG or OEG) deposited on various substrates,such as gold and silicon oxide, have been most extensively studied, andremain the most protein-resistant materials. [Vermette, P.; Meagher, L.“Interactions of phospholipid- and poly(ethylene glycol)-modifiedsurfaces with biological systems: relation to physico-chemicalproperties and mechanisms.” Colloid Surf B-Biointerfaces 2003, 28,153-198. Chen, S. F.; Yu, F. C.; Yu, Q. M.; He, Y.; Jiang, S. Y. “Strongresistance of a thin crystalline layer of balanced charged groups toprotein adsorption.” Langmuir 2006, 22, 8186-8191. Ostuni, E.; Chapman,R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. “A survey ofstructure-property relationships of surfaces that resist the adsorptionof protein.” Langmuir 2001, 17, 5605-5620. Prime, K. L.; Whitesides, G.M. “Self-Assembled Organic Monolayers—Model Systems for StudyingAdsorption of Proteins at Surfaces.” Science 1991, 252, 1164-1167.]Applicant has developed a practical method to prepare OEG-presentingmonolayers on silicon by surface hydrosilylation using theOEG-terminated alkenes (1, FIG. 7) and hydrogen-terminated siliconsurfaces under UV illumination. [Yam, C. M.; Lopez-Romero, J. M.; Gu, J.H.; Cai, C. Z. “Protein-resistant monolayers prepared by hydrosilylationof alpha-oligo(ethylene glycol)-omega-alkenes on hydrogen-terminatedsilicon (111) surfaces.” Chem. Commun. 2004, 2510-2511.] In theresulting monolayers the molecules are bound to the silicon surfacethrough Si—C bonds. Through extensive optimization of thehydrosilylation process as well as the apparatus, the stability of theseOEG-terminated monolayers and their ability to resist non-specificadsorption of proteins were greatly enhanced. The results are summarizedin Table 1. Remarkably, the films remained to be highly resistant toprotein adsorption after 4 weeks in PBS buffer at 37° C. To ourknowledge, these films exhibit the highest stability amongprotein-resisting monolayers reported to date. Furthermore, themonolayers absorbed less than 3% monolayer of proteins in cell culturefor 17 days, also representing the most protein-resistant monolayersreported to date.

TABLE 1 Protein-resistance* of OEG-terminated Monolayers on SiliconAfter Subjected to Various Conditions over Various Periods of Time TimeAdsorption of protein Conditions (days) (% monolayer)** Freshly-madefilms 0 0 PBS buffer at 37° C. 28 0.5 Freshly-made films tested 0 0 withserum at a* D1 cell culture at 37° C. 7 1.2 MC3T3 cell culture at 37° C.17 2.8 *Expressed as the percentage of monolayer of fibrinogen absorbedon the sample after 1 h in 1 mg/mL fibrinogen solution, measured byN_(OEG)/N_(Si—H) × 100 (%) where N_(OEG) and N_(Si—H) are the XPS N 1 sphotoelectron signal intensity of the protein on the OEG films andH—Si(111) surface, respectively. The ellipsometric thickness offibrinogen films on H—Si(111) surface is 6 nm, corresponding to amonolayer of the protein. [Yam, C. M.; Lopez-Romero, J. M.; Gu, J. H.;Cai, C. Z. “Protein-resistant monolayers prepared by hydrosilylation ofalpha-oligo(ethylene glycol)-omega-alkenes on hydrogen-terminatedsilicon (111) surfaces.” Chem. Commun. 2004, 2510-2511.] **The standarddeviations are 15% of the values, since we did not control the substrateorientation during XPS measurement. Wallart, X.; de Villeneuve, C. H.;Allongue, P. “Truly quantitative XPS characterization of organicmonolayers on silicon: Study of alkyl and alkoxy monolayers onH—Si(111).” J. Am. Chem. Soc. 2005, 127, 7871-7878. a* Bovine serumalbumin (1% in PBS) for one day, no protein adsorption (0%) wasdetected.Click Reactions on the Surfaces

To allow highly specific interactions of the monolayers on silicon withbiomolecular targets, we need to incorporate handles on the films fortethering the molecular probes. Although a variety of surfacesfunctional groups have been introduced to monolayers grown byhydrosilylation on silicon surfaces, [Buriak, J. M. “Organometallicchemistry on silicon and germanium surfaces.” Chem. Rev. 2002, 102,1271-1308.] incorporation of functional groups to allow 1,3-dipolarcycloaddition of terminal alkynes and azides (“click reaction”) Kolb, H.C.; Finn, M. G.; Sharpless, K. B. “Click chemistry: Diverse chemicalfunction from a few good reactions.” Angew. Chem.-Int. Edit. 2001, 40,2004-2021. has not be demonstrated. Click reaction is particularlysuitable for bioconjugation since it usually gives high yields underphysiological conditions, and is compatible with a wide range offunctional groups and biomolecules. [Kolb, H. C.; Finn, M. G.;Sharpless, K. B. “Click chemistry: Diverse chemical function from a fewgood reactions.” Angew. Chem.-Int. Edit. 2001, 40, 2004-2021. Brennan,J. L.; Hatzakis, N. S.; Tshikhudo, T. R.; Dirvianskyte, N.; Razumas, V.;Patkar, S.; Vind, J.; Svendsen, A.; Nolte, R. J. M.; Rowan, A. E.;Brust, M. “Bionanoconjugation via click chemistry: The creation offunctional hybrids of lipases and gold nanoparticles.” BioconjugateChem. 2006, 17, 1373-1375. Lee, J. K.; Chi, Y. S.; Choi, I. S.“Reactivity of acetylenyl-terminated self-assembled monolayers on gold:Triazole formation.” Langmuir 2004, 20, 3844-3847. Lin, P. C.; Ueng, S.H.; Tseng, M. C.; Ko, J. L.; Huang, K. T.; Yu, S. C.; Adak, A. K.; Chen,Y. J.; Lin, C. C. “Site-specific protein modification throughCu—I-catalyzed 1,2,3-triazole formation and its implementation inprotein microarray fabrication.” Angew. Chem.-Int. Edit. 2006, 45,4286-4290. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K.B. “A stepwise Huisgen cycloaddition process: Copper(I)-catalyzedregioselective “ligation” of azides and terminal alkynes.” Angew.Chem.-Int. Edit. 2002, 41, 2596-2599. Sun, X. L.; Stabler, C. L.;Cazalis, C. S.; Chaikof, E. L. “Carbohydrate and protein immobilizationonto solid surfaces by sequential Diels-Alder and azide-alkynecycloadditions.” Bioconjugate Chem. 2006, 17, 52-57. Tornoe, C. W.;Christensen, C.; Meldal, M. “Peptidotriazoles on solid phase:[1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolarcycloadditions of terminal alkynes to azides.” J. Org. Chem. 2002, 67,3057-3064. Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless,K. B.; Finn, M. G. “Bioconjugation by copper(I)-catalyzed azide-alkyne[3+2] cycloaddition.” J. Am. Chem. Soc. 2003, 125, 3192-3193. Tointroduce the alkynyl groups to the surfaces, we employed the OEG-alkeneterminated with a trimethylsilyl protected ethynyl group (2, FIG. 7) toprepare mixed monolayers with the matrix molecule 1 by hydrosilylationon H—Si(111) surfaces under UV illumination. To demonstrate the presenceof ethynyl groups on the surface, we performed click reaction on themixed monolayers using the azide 3 tethering a perfluorooctyl chain. XPSshowed a strong F 1s signal upon this reaction but no F 1s signal uponsubjecting the above films to the same conditions except the absence of3. This result suggests the presence of ethynyl groups that couldundergo click reactions.

To demonstrate the potential biological applications of theethynyl-presenting OEG surfaces on silicon substrates, we “clicked”mannose onto the surface using the azide 4 (FIG. 7). The bacteria usedin the test were E. coli (strain 83972), E. coli (strain BL21) andPseudomonas aeruginosa (strain 19660). All bacteria did not absorb onthe OEG surfaces derived from 1 (FIG. 8 a). The mannose-presentingsurfaces could indeed capture E. coli (strain 83972) as shown by thefluorescence images (FIG. 8 b), while it could not capture the other twobacteria that are lack of mannose acceptors.

Single Molecule Arrays for Study of Cell Responses

Several nanopatterning techniques have been developed for fabricatingprotein nanoarrays, including dip-pen nanolithography, nanografting,self-assembly of polymer micelles with a metallic core, and E-beamlithography. [Blattler, T.; Huwiler, C.; Ochsner, M.; Stadler, B.;Solak, H.; Voros, J.; Grandin, H. M. “Nanopatterns with biologicalfunctions.” J. Nanosci. Nanotechnol. 2006, 6, 2237-2264.] Alternatively,we have developed a method based on conductive AFM (cAFM)nanolithography on OEG-terminated monolayers on silicon. We havedemonstrated the highest resolution (25 nm) for fabrication of proteinnanoarrays on protein-resistant surfaces. Gu, J. H.; Yam, C. M.; Li, S.;Cai, C. Z. “Nanometric protein arrays on protein-resistant monolayers onsilicon surfaces.” J. Am. Chem. Soc. 2004, 126, 8098-8099.

To gain insights of the cell responses to nanoscale presentation ofsignaling molecules, we are developing a methodology to preciselycontrol the location and number of ligands on the surface as shown inFIG. 9. Briefly, nanotemplates presenting carboxylic acid groups aregenerated on OEG-terminated surfaces by cAFM, which employs a biasvoltage to induce local electrochemical oxidation on the monolayer. Thefeature size of the pattern should be small enough to accommodate nomore than one generation 5 polyamidoamine (PAMAM) molecule, which ispartially coated with OEG and modified with a defined number offunctional groups such as ethynyl or azido groups for click reactions.Upon amidation of the functionalized PAMAM on the nanotemplate,biomolecular probes can be tethered to the templates via clickreactions.

To realize this concept, a crucial step is to reduce the feature sizedown to about 10 nm, which is similar to the size of one generation 5PAMAM molecule spreading on polar surfaces. Through mechanistic study ofthe cAFM process using model compounds, we found that it is possible toselectively oxidize only the surfaces of the OEG monolayer, and thefeature size of the generated patterns is determined by the size ofwater meniscus where hydroxy radicals are generated. After extensiveoptimization of the conditions for c-AFM, we successfully reduced thefeature size down to about 10 nm.

We have demonstrated that OEG-terminated monolayers prepared by ourmethod are among the most protein-resistant and stable monolayersreported to date. The films absorbed less than 3% monolayer of proteinafter 17 days in MC3T3 cell culture at 37° C., and remainedprotein-resistant even after 28 days in PBS buffer. We are collaboratingwith other groups to perform implantation of these OEG-coated siliconsamples to study their in vivo biocompatibility and long-term stability.We also prepared mixed OEG-monolayers presenting ethynyl groups ontoH—Si (111) surfaces, and introduced mannose groups onto the surfaceusing click reactions. Finally, we showed that the mixed monolayerspresenting mannose selectively captured E. coli that has mannosereceptors. We will collaborate with other groups to fabricate demodevices using the above platforms for various biosensing applications.

In contribution to our effort to prepare single molecule arrays, aftermechanistic studies and extensive optimization of the c-AFMnanopatterning on the OEG monolayers, we have successfully reduced thefeature size down to ˜10 nm, which could accommodate no more than onelarge PAMAM molecule on each spot. Future work will includedemonstration of tethering of single PAMAM molecule onto each spot onthe nanopattern and the use of these single molecule arrays to studycellular pattern-recognition.

STILL FURTHER EXAMPLES

Modification of silicon substrates with biomolecules is of tremendousinterest for the development of silicon-based biodevices such as(implantable) biosensors and neuron interfaces. [Yang, W. S.; Butler, J.E.; Russell, J. N.; Hamers, R. J. Analyst 2007, 132, 296; K'Owino, I.O.; Sadik, O. A. Electroanalysis 2005, 17, 2101; Hochberg, L. R.;Serruya, M. D.; Friehs, G. M.; Mukand, J. A.; Saleh, M.; Caplan, A. H.;Branner, A.; Chen, D.; Penn, R. D.; Donoghue, J. P. Nature 2006, 442,164.] Using silicon as the substrates for these applications has severalunique advantages, including the availability of well-establishedmicro-fabrication processes and electronic and mechanical properties.For these applications, it is often desirable to modify silicon surfacesto reduce inflammatory response that degrades their performance. Toaddress this need, we have developed a practical technique [Cai, C.;Yam, C. M.; Xiao, Z.; Gu, J. Modification of silicon-containing scanningprobe microscopy tips and growth of oligo- or poly(ethylene glycol)films on silicon surfaces through formation of Si—C bonds; U.S. Pat. No.7,247,384; Yam, C. M.; Lopez-Romero, J. M.; Gu, J. H.; Cai, C. Z. Chem.Commun. 2004, 2510; Yam, C. M.; Gu, J.; Li, S.; Cai, C. J. ColloidInterface Sci. 2005, 285, 711.] for preparing highly protein-resistantand stable monolayers by hydrosilylation [Linford, M. R.; Fenter, P.;Eisenberger, P. M.; Chidsey, C. E. D. J. Am. Chem. Soc. 1995, 117, 3145;Buriak, J. M. Chem. Rev. 2002, 102, 1271.] of the alkene 1 onhydrogen-terminated silicon surfaces. The monolayers consist of anoligo(ethylene glycol) (OEG) layer on top of an ultrathin (13 Å),well-ordered, highly dielectric alkyl layer directly bound to thesilicon surface. These monolayers are bound to the silicon surface viaSi—C bonds with a high density, rendering them the mostprotein-resistant and stable monolayers reported to date. In addition,such ultrathin (˜4 nm) monolayers on non-oxidized silicon are idealinterfaces for highly sensitive transduction of electrical andbiological signals. To permit specific interactions with targetedbiological entities, the monolayer surface needs to be functionalizedwith ligands. For this purpose, we are interested in the bioconjugationchemistry based on cycloaddition of acetylenes and azides (“click”reaction), since the chemistry is specific and compatible with a widerange of biomolecules under physiological conditions, [Rostovtsev, V.V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Edit.2002, 41, 2596; Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.2002, 67, 3057; Wu, P.; Fokin, V. V. Aldrichimica Acta 2007, 40, 7;Nandivada, H.; Jiang, X. W.; Lahann, J. Adv. Mater. 2007, 19, 2197;Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R.ACS Chem. Biol. 2006, 1, 644; Lutz, J. F. Angew. Chem. Int. Ed. 2007,46, 1018; Wang, Q.; Chittaborina, S.; Barnhill, H. N. Lett. Org. Chem.2005, 2, 293; Binder, W. H.; Sachsenhofer, R. Macromol. Rapid Commun.2007, 28, 15.] and it has been used to modify various types organic thinfilms. [Ciampi, S.; Bocking, T.; Kilian, K. A.; James, M.; Harper, J.B.; Gooding, J. J. Langmuir 2007, 23, 9320; Lin, P. C.; Ueng, S. H.; Yu,S. C.; Jan, M. D.; Adak, A. K.; Yu, C. C.; Lin, C. C. Org. Lett. 2007,9, 2131; Lin, P.-C.; Ueng, S.-H.; Tseng, M.-C.; Ko, J.-L.; Huang, K.-T.;Yu, S.-C.; Adak, A. K.; Chen, Y.-J.; Lin, C.-C. Angew. Chem. Int. Ed.2006, 45, 4286; Sun, X. L.; Stabler, C. L.; Cazalis, C. S.; Chaikof, E.L. Bioconjugate Chem. 2006, 17, 52; Zhang, Y.; Luo, S.; Tang, Y.; Yu,L.; Hou, K.-Y.; Cheng, J.-P.; Zeng, X.; Wang, P. G. Anal. Chem. 2006,78, 2001; Duckworth, B. P.; Xu, J. H.; Taton, T. A.; Guo, A.; Distefano,M. D. Bioconjugate Chem. 2006, 17, 967; Lummerstorfer, T.; Hoffmann, H.J. Phys. Chem. B 2004, 108, 3963; Lee, J. K.; Chi, Y. S.; Choi, I. S.Langmuir 2004, 20, 3844; Gallant, N. D.; Layery, K. A.; Amis, E. J.;Becker, M. L. Adv. Mater. 2007, 19, 965; Collman, J. P.; Devaraj, N. K.;Eberspacher, T. P. A.; Chidsey, C. E. D. Langmuir 2006, 22, 2457;Fleming, D. A.; Thode, C. J.; Williams, M. E. Chem. Mat. 2006, 18, 2327;Rozkiewicz, D. I.; Janczewski, D.; Verboom, W.; Ravoo, B. J.; Reinhoudt,D. N. Angew. Chem. Int. Ed. 2006, 45, 5292; Devadoss, A.; Chidsey, C. E.D. J. Am. Chem. Soc. 2007, 129, 5370; Bryan, M. C.; Fazio, F.; Lee,H.-K.; Huang, C.-Y.; Chang, A.; Best, M. D.; Calarese, D. A.; Blixt, O.;Paulson, J. C.; Burton, D.; Wilson, I. A.; Wong, C.-H. J. Am. Chem. Soc.2004, 126, 8640; Rohde, R. D.; Agnew, H. D.; Yeo, W. S.; Bailey, R. C.;Heath, J. R. J. Am. Chem. Soc. 2006, 128, 9518; Prakash, S.; Long, T.M.; Selby, J. C.; Moore, J. S.; Shannon, M. A. Anal. Chem. 2007, 79,1661; Nandivada, H.; Chen, H. Y.; Bondarenko, L.; Lahann, J. Angew.Chem. Int. Ed. 2006, 45, 3360; White, M. A.; Johnson, J. A.; Koberstein,J. T.; Turro, N. J. J. Am. Chem. Soc. 2006, 128, 11356; O'Reilly, R. K.;Joralemon, M. J.; Wooley, K. L.; Hawker, C. J. Chem. Mat. 2005, 17,5976; Li, H. M.; Cheng, F. O.; Duft, A. M.; Adronov, A. J. Am. Chem.Soc. 2005, 127, 14518.] Herein, we report a method for incorporatingacetylene derivatives onto well-defined, OEG-based monolayers on siliconsubstrates that allows direct tethering of biomolecules via clickchemistry.

Click reactions have been implemented on various types of substrates,such as gold, [Collman, J. P.; Devaraj, N. K.; Chidsey, C. E. D.Langmuir 2004, 20, 1051; Collman, J. P.; Devaraj, N. K.; Eberspacher, T.P. A.; Chidsey, C. E. D. Langmuir 2006, 22, 2457; Lee, J. K.; Chi, Y.S.; Choi, I. S. Langmuir 2004, 20, 3844.] silicon oxide, [Lummerstorfer,T.; Hoffmann, H. J. Phys. Chem. B 2004, 108, 3963; Rozkiewicz, D. I.;Janczewski, D.; Verboom, W.; Ravoo, B. J.; Reinhoudt, D. N. Angew. Chem.Int. Ed. 2006, 45, 5292; Sun, X. L.; Stabler, C. L.; Cazalis, C. S.;Chaikof, E. L. Bioconjugate Chem. 2006, 17, 52.] graphite, [Senyange,S.; Anariba, F.; Bocian, D. F.; McCreery, R. L. Langmuir 2005, 21,11105.] and porous silicon. [Bateman, J. E.; Eagling, R. D.; Worrall, D.R.; Horrocks, B. R.; Houlton, A. Angew. Chem. Int. Ed. 1998, 37, 2683.]Only a few examples were reported on non-oxidized silicon surfaces.[Hurley, P. T.; Ribbe, A. E.; Buriak, J. M. J. Am. Chem. Soc. 2003, 125,11334; Rohde, R. D.; Agnew, H. D.; Yeo, W. S.; Bailey, R. C.; Heath, J.R. J. Am. Chem. Soc. 2006, 128, 9518; Ciampi, S.; Bocking, T.; Kilian,K. A.; James, M.; Harper, J. B.; Gooding, J. J. Langmuir 2007, 23,9320.] Direct bonding on non-oxidized silicon eliminates the dielectricsilicon oxide layer, thus greatly improving the electrical communicationwith the silicon substrate.

Initially, we attempted to perform photo- or thermal-initiatedhydrosilylation with alkenes terminated with an azido group on H—Si(111)surfaces, but failed, likely due to the instability of the azido groupsunder the deposition conditions. We then envisioned that ethynyl groupsmight survive the reaction conditions if they were protected with abulky group such as trimethylsilyl (TMS) group. Although thepropiolamide derivative 2 (FIG. 9) polymerized under UV, thenon-conjugated alkyne 3 was stable and completed hydrosilylation onhydrogen-terminated Si(111) surfaces within 2 h under UV (254 nm). Theresulting monolayers exhibited an ellipsometric thickness of 52.3±1.2 Å,close to the length (59 Å) of extended 3 calculated by MM2. Thepercentage of C—O moieties in the film was 56.5% (expected: 58%) asdetermined by X-ray photoelectron spectroscopy (XPS) by deconvolutingthe C 1s signals at 286.7 eV (assigned to C—O) and at 285.0 eV (assignedto the rest C atoms).² However, the attempted desilylation followed byclick reaction on the monolayers was not successful, probably becausethe removal of the TMS group was problematic (see below). To allowreliable monitoring of the deprotection step, we used a fluoratedalkylsilyl group to protect the terminated alkyne as in 4. Surfacehydrosilylation of 4 resulted in a film with an ellipsometric thicknessof 60.5±0.9 Å, close to the calculated length (62 Å) of the extended 4.According to the XPS measurement, the (C—O) % in the film was 54.1%(expected: 54.9%), and the C/O/F ratio was 1:0.28:0.027, close to theexpected value of 1:0.27:0.023. Removal of the fluorinated alkylsilylgroup on the monolayer was monitored by the F 1s XPS signal. In contrastto the rapid desilylation (completed within 30 min) for compound 4 inTHF solution of Bu₄NF (0.2 equiv.), desilylation of the monolayersderived from 4 in the same solution was sluggish, probably due to thecombination of steric hindrance and hydrophobicity of the surface.Indeed, for the mixed monolayers prepared by co-deposition of 1 and 4 ina 10:1 ratio, the desilylation proceeded in a substantially higher rate,especially in the presence of Cu⁺ that coordinates with C≡C and thusactivates the nucleophilic attack on Si. Thus, the desilylation wascompleted in 40 min in a solution of Bu₄NF (0.3 mM) and CuOAc (0.03 mM)in THF. Unfortunately, under these conditions partial oxidation of thesilicon interface also occurred, as shown by the appearance of thesilicon oxide peak at 103 eV.

In search for a protecting group for terminal alkynes that could beremoved under very mild, neutral conditions, we turned our attention totrimethylgermanyl (TMG) group. [Ernst, A.; Gobbi, L.; Vasella, A.Tetrahedron Lett. 1996, 37, 7959; Cai, C. Z.; Vasella, A. Helv. Chim.Acta 1995, 78, 732.] TMG group on terminal alkynes can be readilyremoved in protic solvents in the presence of catalytic amounts of Cu⁺.The monolayer presenting C≡C-TMG groups was derived from the alkenyne 5(FIG. 9). The ellipsometric thickness of the monolayer was 58.4±0.1 Å,close to the estimated length (59 Å) of the extended molecule,indicative of no hairpining of the alkyne molecule on the surface andthat the observed film is indeed a monolayer thick. The result isfurther verified through the appearance of the underlying atomic stepsof the silicon (111) substrate as shown on the AFM image, as shown inFIG. 10.

XPS spectra acquired on the TMG terminated monolayer are shown on FIG.11. The survey spectra showed the presence of C, O, Ge and Si, asexpected. High resolution scans of the Si 2p spectra showed the absenceof peak at 101-104 eV regions suggesting no significant oxide orsuboxide silicon was present. This result is also in accord with theobserved formation of high density monolayer from the TMG-terminatedalkene 5. XPS result on the Ge 3d region showed a strong peak at 30.1eV, indicating the presence of the TMG group on the surface. Anadvantage of using TMG as the protecting group is that the Ge 3d signalcan be used to estimate the density of the ethynyl groups on thesurface, and the yield of the deprotection. The narrow scan in the C 1sregion showed the presence of C—C, C—O peaks with a mean binding energyat 285.0 eV and 286.7 eV, respectively. The peaks were analyzed first bybackground subtraction using the Shirley routine and a subsequentnon-linear fitting to mixed Gaussian-Lorentzian functions. The data fromthe C1s spectra were fitted to functions having 80% Gaussian and 20%Lorentzian character. A satellite peak shifted at a slightly lowerbinding energy (283.8 eV) was also assigned to contributions from C—Si.This is consistent with the expected result for a slightly negativelycharged carbon atoms (Si—C—R₁, R₂, R₃; R₁, R₂, R₃═C, H). Atomicconcentration contributions were also determined from the XPS and theratio of concentrations of C:O:Ge was found to be 1:0.30:0.029 and isclose to the expected value of 1:0.29:0.026.

As expected, 95% of the TMG group on the monolayer was removed in 1 h ina solution of Cu(MeCN)₄ PF₆ (10 mM) and ascorbic acid (50 mM) inmethanol under nitrogen. The click reaction on the deprotected alkynesurface was tested using the fluoride-containing azide 6 (5 mM) andCu(MeCN)₄ PF₆ (10 mM) in methanol/ethanol/water (v/v/v 2:1:1) overnight.XPS showed the F 1s signal at 690 eV and N 1s signal at 401 eV,indicating the presence of the CF₃ group, and the triazole and amidegroups, respectively. No signal was present at 405 eV corresponding tothe central, electron-deficient N-atom in the azido group, indicating nophysisorption of 6 in the film.

An additional advantage of using TMG protecting group is that theCu⁺-catalyzed deprotection proceeds faster than the click reaction underthe same conditions. Hence, they can be combined into one step (Scheme1). Indeed, XPS data showed that the monolayer presenting C≡C-TMG groupsunderwent click reaction with the azide 6 (5.0 mM) in the presence ofCu(MeCN)₄ PF₆ (2.5 mM) and ascorbic acid (25.0 mM) overnight. The F 1ssignal appeared at 690 eV and N 1s at 401 eV, accompanied by thereduction of the Ge 3d signal intensity by 95% (FIG. 12). The N 1ssignal was deconvoluted and fitted to three peaks assigned to CONH(401.7 eV), N—N═N (400.8 eV), and N—N═N (400.1 eV); the ratio of thepeak areas was about 1.2:2:1. Moreover, the N/F ratio was 1.31 (expected1.33). Based on the C/F ratio (32.11) measured by XPS, the reactionyield was estimated to be 43%. The incomplete click reaction between thesurface alkynes and the OEG-azide 6 in the solution remained unclear. Itmight have been due to the steric hindrance, or homo-coupling of theterminal alkynes in the presence of Cu⁺ and adventitious O₂.

Another example of surface click reaction is described below. In areaction vial containing the alkyne terminated surface, 200 μL of thecopper catalyst (5 mM) and ascorbic acid (50 mM) in methanol was added.The solution was allowed to stand for 10 minutes and was followed by theaddition of the azide (10 mM in ethanol/water (1:1 v/v)). Thecycloaddition reaction was allowed to proceed for 12 h under N₂environment. FIG. 13 shows the XPS survey spectra and high resolutionspectra before and after the click reaction. The survey spectra revealthe presence of the N1 s and F1s signals, which were not observed beforethe click reaction. The strong peak at 690 eV from the F1 s spectrasuggests the presence of CF₃ group, while the high resolution N1 sspectra provided evidence for the formation of triazole moiety and thusthe occurrence of click. Narrow scan of the Si 2p region showed nodetectable levels of SiOx species in the 102-104 eV region (FIG. 14).The N1 s spectra (FIG. 13) was deconvoluted and fitted into three peaks.The peaks were assigned as the amide nitrogen (402.1 eV), the doublebonded nitrogen (401.2 eV) in the triazole ring and the singly bondednitrogen (400.5 eV) in the ring. The ratio of the integrated peaks werefound to be 1:2:1 and is consistent with the expected structure.Moreover, no peak was observed at ˜405 eV, corresponding to the electrondeficient nitrogen group in the azide group. This signifies the absenceof any unreacted azide present in the monolayer.

The topographies of the monolayer were observed using AFM (FIG. 15) andreveals a flat and homogeneous surface even after the coupling reaction.The Si (111) steps were also visible suggesting a monolayer formation.The occurrence of a monolayer after the click reaction was furtherverified with the observed increase in thickness. The thickness wasfound to be 68.4±0.4 Å which is close to its calculated thickness 70 Å.

Optimization of the Yields

The Influence of Oxygen

The CuAAC reactions were performed under different conditions withvaried oxygen content. Two sets of experiments were performed and thegeneral procedure mentioned with respect to FIG. 13 was followed. Theresults are summarized in Table 1. Higher yields were observed onanaerobic conditions and a significantly low yield for click systems ranwithout the exclusion of air (˜22% versus ˜53%). The results areattributed to the high sensitivity of Cu⁺ towards oxidation by O₂ toform Cu²⁺, even in the presence of ascorbic acid that reduces Cu²⁺ backto Cu⁺. Upon oxidation, the copper catalyst is deactivated and theresultant to Cu²⁺ species promote the homocoupling of alkynes.

Given that higher yields were observed for click reactions performedunder anaerobic conditions, coupling reactions for all succeedingconditions were performed under anaerobic environment. Methanol, thesolvent used for dissolving the catalyst was freeze-thawed several times(at least 6×) prior to each use. The rest of the conditions were keptconstant, except that the concentration of the copper complex wasvaried. Referring to FIG. 16, conditions were: 200 μL Cu(MeCN)₄ PF₆(0.25, 2.5, 5, 7.5, 10, 15 mM) and ascorbic acid (50 mM) in methanol;250 μL azide (10 mM) in ethanol/water (1:1 v/v); reaction time: 12 h;environment: anaerobic. The results are summarized in FIG. 16, showingthat increasing the concentration of the catalyst lead to a higheryield. However, with higher loading of Cu-catalyst, more Cu²⁺ weredetected to remain on the film after the reaction. We found that theoptimum Cu concentration was 2.5 mM. Although this concentration did notprovide the highest yield, Cu²⁺ were not detected on the surface, whichis needed for bioapplications since copper is cytotoxide and catalyzesthe degradation of the organic films.

Using the best concentration of Cu complex and running the reactionunder controlled atmosphere. The effect of the reaction time on theyield was investigated (Table 2). Two sets of experiments were performedat varying reaction time (3, 6, 12 and 18 h), in the presence of Cucatalyst and ascorbic acid. Satisfactory yields (>65%) were obtainedonly after 12 h. The reaction stopped after ˜70% conversion.

The effect of using a chelating agent to sequester Cu²⁺ in the surfaceon the yield of the coupling reaction was considered. EDTA is a wellknown chelating agent of heavy metals such as Cu²⁺.

Two sets of measurements were performed to test the effect of adding achelating agent in the reaction mixture (Table 3). In the firstmeasurement, EDTA was added into the reaction mixture after 6 h ofcoupling. After the addition, the films were kept into the reactionvessel for another 6 h. It was mentioned from previous results that thereaction has began after 6 h though the reaction has not reached itscompletion. The results for the first case with EDTA showed a yield of˜46%, which is similar to the yield that was obtained for a 6 h reactionwithout EDTA. On the other hand, no reaction was observed after 12 h ifEDTA was added at the beginning. Significantly, the addition of EDTAafter the reaction greatly decreased the amount of Cu left in the film,as shown in the narrow Cu 2p scan before and after EDTA addition (FIG.17).

Using the Ligands for the Surface CuAAC Reaction.

Referring to FIG. 18, the structure of the ligand that was used forclick reaction is composed of triazole ring, an amine functionality andEG units. Conditions for the ligand based click: 200 μL Cu(MeCN)₄ PF₆(2.5 mM) and ascorbic acid (50 mM) in methanol; 250 μL azide (10 mM) inethanol/water (1:1 v/v); 200 μL ligand 28 (25 mM); reaction time: 1, 2,4, 15 h. Two sets of experiments were performed at reaction times of 1,2, 4 and 15 h in the presence of the ligand 8 (FIG. 18). Evidently, theligand accelerated the rate of the Cu⁺ dependent process for couplingreactions interrupted after 1 h. The extent of conversion after 1 and 2h interruptions was found to be ˜19 and ˜57%, respectively. The resultsshowed a significant improvement in the yield as compared to the noreaction observed for a typical “ligand free” system interrupted after 3h. Upon a 4 h cycloaddition reaction, ˜75% of the alkyne terminatedmonolayer was converted to the corresponding triazole product. Whenprolonged reaction time was employed (15 h) no significant benefitsassociated with the presence of the ligand was evident.

To this end, the 4 h reaction with the ligand showed the best result forthe cycloaddition reaction of EG containing alkyne and azidefunctionalized reacting species. A plot comparing the percent conversionof the click reaction as function of reaction time in the presence andabsence of ligand depicts the remarkable improvement of the couplingyield (from 69 to 75%). More importantly, the incorporation of theligand, greatly advanced the potential of click reaction in the surfaceby reducing the time of reaction by 33%. Furthermore, the amount ofcatalyst

Demonstration of the Selective Detection of E. Coli Using the Scheme forBiofunctionalization of Silicon Surfaces

Reaction conditions were optimized (FIG. 19) for tethering the azido-OEGmannoside 7 and the azido-OEG glucose 9 onto the above monolayerspresenting CC-TMG groups. To demonstrate the specific detection ofbacteria using the functionalized surfaces, several modifications of theEscherichia coli strain 83972 [Andersson, P.; Engberg, I.; Lidinjanson,G.; Lincoln, K.; Hull, R.; Hull, S.; Svanborg, C. Infect. Immun. 1991,59, 2915.] were created for the study by Dr. Barbara W. Trautner atBaylor College of Medicine. In particular, E. coli Hu2545 overexpressingmannose-binding type 1 fimbriae [Pratt, L. A.; kolter, R. Mol.Microbiol. 1998, 30, 285.] by transforming E. coli 83972 with pSH1 (an18 copy plasmid with the intact fim operon) were created. [Hull, R. A.;Gill, R. E.; Hsu, P.; Minshew, B. H.; Falkow, S. Infect. Immun. 1981,33, 933.] E. coli Hu2634 is an 83972 mutant lacking fim, created bytransforming E. coli 2222 (83972 ΔfimHΔpapG) with pACYC184ΔTc. [Chang,A. C. Y.; Cohen, S. N. J. Bacteriol. 1978, 134, 1141.] Both strains weretransformed with pGreen so that they fluoresced green. The mannose- andglucose presenting films and a film of 5 as control were incubated for 7hours in Luria Bertani media containing either Hu2545 or Hu2634. Asexpected, the fim+ E. coli strain Hu2545 attached to themannose-presenting surfaces, while the fim− strain Hu2634 did not adhereto the surfaces (FIG. 20). Both strains did not attached to theglucose-presenting surfaces. The contrast is extremely high.Furthermore, no E. coli Hu2545 were found on the film derived from 5;the fluorescence images (not shown) were similar to FIG. 20 b. Referringto FIG. 20, the films were prepared by hydrosilyation of 5 on H—Si(111)and then click reaction with 7.

Applicant has shown that TMG-protected α,ω-alken-ynes undergo selectivesurface hydrosilylation on H-terminated silicon. Click reactions can bedirectly performed on the resulting monolayers presenting C≡C-TMGgroups. Biomolecules can be tethered onto OEG monolayers on siliconusing this method, as demonstrated by the specific adherence of E. coliexpressing mannose-binding fimbriae onto the mannose-presentingsurfaces.

Development of High Efficient Copper Catalysts for Bioconjugation ontoSurfaces

Despite its popularity, there are a few drawbacks of click reactions(more accurate term is Cu-catalyzed azide-alkyne 1,3-dipolarcycloaddition (CuAAC)) for bioconjugation. First, Cu(I) catalyst isthermodynamically unstable and can be easily oxidized by oxygen in airand/or disproportionate in aqueous solution to catalytically inactiveCu(II). Reducing agents, especially ascorbic acid, are commonly used toreduce Cu(II) back to Cu(I). However, Cu(I)/ascorbate mediateddegradation of the biological scaffolds has been observed. Second, Cu(I)is highly cytotoxic, thus greatly limiting the CuAAC reaction forbioconjugation in the presence of live cells. Third, the CuAAC reactionis still not a true click reaction: it is quite slow, and the yields aresometimes quite low. The objective of this project is to develop coppercatalysts and conditions to allow the CuAAC reactions to completerapidly (within a few minutes) in almost quantitative yield using a lowconcentration (<10 μM) of Cu(I) catalyst under philological conditions.

Recently, the Sharpless group has reported the tris(triazo) ligand 1that significantly accelerates the CuAAC reaction and stabilizes the +1oxidation state of the copper catalyst. Its tetradentate binding abilityis believed to protect the Cu(I) center against destabilizinginteractions. The tertiary amine and the [1,2,3]-triazolefunctionalities likely work in concert to promote the catalyticefficiency of Cu(I): the sterically encumbered N-atom of the tertiaryamine accelerates the reaction by increasing the electron density on themetal center, while the weaker triazole ligand comes off the coppercenter temporarily to allow the formation of the Cu(I)-acetylidecomplex, which is then carried through the catalytic cycle. This ligandis now commercially available, and has been widely used in CuAACreactions performed in organic solvents, although using it forbioconjugation involving nucleic acids, proteins and cells is limited.

Despite of its wide use, the tris(triazole) 1 (FIG. 21) is hardlysoluble in water, thus limiting its utility in bioconjugation reactions.We reasoned that tethering the amphiphilic oligo(ethylene glycol) (OEG)groups to 1 would not only render its Cu(I) complex water-soluble, butalso improve its biocompatibility. The synthesis of the OEG-modifiedligand 2 (FIG. 21) was straightforward. Thus, monosubstitution of1,4-bis(chloromethyl)benzene (3) with methyl tetra(ethylene glycol) 4provided the OEG-benzyl chloride 5 in 77% yield. The azido group wasreadily introduced to 5 in 90% yield. 1,3-Dipolar cycloaddition of theresultant azide 6 with tripropargyl amine (7) in the presence of Cu(I)afforded the water soluble tris(triazole) ligand 2 in good yield.

To test the efficiency of the ligand 2, several azido derivatizedbiomolecules were synthesized (FIG. 22). Protected tetrapeptide GRGD 11was attached azido modified oligoethylene glycol 12 via carbodiimidecoupling to give 13 which on deprotection gave 14.

Similarly acetate protected bromide derivative of glucose 15 wasattached to azido modified oligoethylene glycol 16 to give 17 which ondeprotection gave azido modified glucose 18. On the same line biotin-NHS19 was attached with azido modified oligoethylene glycol 20 to giveazido modified biotin. Florescent dye 8 was coupled with hexynoic acid(9) via carbodiimide chemistry to give alkyne terminated dye 10.

The CuAAC reactions of these azides with in the presence of the ligand 2in aqueous medium under ambient conditions to give products 22, 23 and24 in excellent yields (FIG. 22).

Although the tris(triazo) ligands 1 and 2 greatly accelerate the rate ofCuAAC under ambient conditions with excellent yields, the reaction isstill relatively slow (>12 hours for completion, FIG. 23). In addition,we observed that 2 did not promote the CuAAC reaction on monolayerspresenting ethynyl groups, probably due to the steric hindrance. Finnand other groups have reported more efficient ligands for CuAACreaction, especially the water soluble bathophenanthroline andBenzimidazole. The rate enhancement of these ligands is apparentlyattributed to the electron-donating amino groups. Aliphatic amines andpyridine derivatives have been used as ligands of Cu(I) and/or proticbases to enhance the CuAAC reaction, but the results are often notsatisfactory. Unfortunately, the stronger electron-donating ability ofthe ligands also render their Cu(I) complexes extremely sensitive tooxygen. To solve this problem, we used Oxyrase to remove oxygen in thesolution. Oxyrase is prepared from Enterococcus coli, and containsenzymes that catalyze the reduction of O₂ in water in the presence of ahydride source such as lactic acid.

To screen these ligands, click-activated fluorogenic labeling techniquewas used. This method was reported by wong et. al. involves the use of1,8-naphthalimide derivative 30 (FIG. 25) designed for click-activatedfluorescence with an azide moiety attached at the 4 position.

We modified the reported probe to be water soluble 31 (FIG. 25) so thatrate of click chemistry could be followed in aqueous media. The watersoluble azido modified 1,8-naphthalimide derivative has a absorptionmaxima at 355 nm and emission at 422 nm. The increase of florescenceover time indicates the rate of reaction. Under ambient conditions OEGderivative of TBTA 2 performed the best but under anaerobic conditionligand 28 (FIG. 24) was found to perform better.

In our system, the ligand that we have used has a triazole ring, anamine functionality and an EG units. The ring stabilizes the Cu⁺ byforming a complex, which encapsulates Cu thereby preventing it fromoxidizing. The additional N, which is not from the ring donates e-s toCu thus making it more labile and increases the rate of reaction. The EGfunctionality was incorporated to increase its solubility in watermaking it biocompatible.

EXPERIMENTAL SECTION

General Information:

Air sensitive reactions were performed under a nitrogen atmosphere usingSchlenk technique. All reagents were purchased from Sigma-Aldrich orAlfa Aesar, and used without purification. Flash chromatography wascarried out on silica gel (60 Å, Sorbent Technologies). All ¹H— and¹³C-NMR spectra were recorded in CDCl₃ using residual CHCl₃ as internalstandard. Mass spectroscopy (MS) measurement was carried out usingelectrospray ionization (ESI) technique. Usual workup procedure: Thereaction was quenched with water, and the mixture was extracted with3×CH₂Cl₂. The organic layers were combined, washed with water and brine,dried over Na₂SO₄ or Mg₂SO₄, and filtered.

A soln. of S2¹ (317 mg, 1.0 mmol) in dry THF (3 mL) was added to NaH (18mg, 0.75 mmol) under N₂. After stirring for 4 h, the mixture was treatedwith a soln. of S1² (375 mg, 0.75 mmol) in dry THF (2 mL), and stirredfor 48 h at room temperature. Usually workup and flash chromatography(ethyl acetate/methanol=30/1) provided S3 as a colorless oil (184 mg,58% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.16-1.31 (m, 12H), 1.47-1.54 (m,2H), 1.95-2.02 (m, 2H), 3.32-3.41 (m, 4H), 3.50-3.83 (m, 38H), 4.86-4.97(m, 2H), 5.69-5.80 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 25.9, 28.8, 28.9,29.26, 29.29, 29.4, 29.5, 33.6, 50.5, 69.87, 69.90, 70.42, 70.47, 70.52,70.54, 71.4, 113.9, 139.0. ESI-MS: m/z 658 [M+Na]⁺.

To a soln. of S11 (0.8 g, 2.3 mmol) in dry THF (10 mL) was added NaH(200 mg, 5.0 mmol, 57-63% in oil) under nitrogen at 0° C. The mixturewas stirred for 1 h, treated with a soln. of S8 (1.1 g, 2.3 mmol) in dryTHF (10 mL), and stirred at room temperature overnight. The reaction wasquenched by water. The mixture was extracted with CH₂Cl₂ (3×30 mL). Theorganic layers were combined, washed with water (30 mL) and brine (30mL), dried over Na₂SO₄, and filtered. The residue was purified by flashchromatography (ethyl acetate/methanol=20/1) to give S12 (1.1 g, 72%yield) as light yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 1.28-1.38 (m,10H), 1.54-1.62 (m, 2H), 1.79 (tt, J=6.0, 6.6 Hz, 2H), 1.94 (t, J=2.7Hz, 1H), 2.03 (q, J=6.6 Hz, 2H), 2.28 (dt, J=2.7, 6.9 Hz, 2H), 3.44 (t,J=6.9 Hz, 2H), 3.53-3.70 (m, 42H), 4.90-5.02 (m, 2H), 5.74-5.87 (m, 1H).¹³C NMR (75 MHz, CDCl₃) δ 15.0, 25.9, 28.3, 28.7, 28.9, 29.2, 29.4,33.6, 68.3, 69.3, 69.9, 70.0, 70.35, 70.40, 71.3, 83.7, 113.9, 139.0.ESI-MS: m/z 685 [M+Na]⁺.

At −78° C., n-butyllithium (2.2 M in hexane, 1.1 mL, 2.4 mmol) was addedto a solution of S12 (200 mg, 0.3 mmol) in dry THF (20 mL) in thepresence of 3 Å MS (20 mg). After stirring for 1 h at this temperature,chlorotrimethylsilane (261 mg, 2.4 mmol) was added. The reaction mixturewas warmed to room temperature and stirred overnight. The reactionmixture was purified by flash chromatography (ethylacetate/methanol=100/3) to afford 3 (150 mg, 68% yield) as light yellowoil. ¹H NMR (300 MHz, CDCl₃) δ 0.14 (s, 9H), 1.25-1.41 (m, 10H),1.51-1.62 (m, 2H), 1.75-1.82 (m, 2H), 2.00-2.08 (m, 2H), 2.31 (t, J=6.6Hz, 2H), 3.44 (t, J=6.6 Hz, 2H), 3.52-3.71 (m, 42H), 4.90-5.01 (m, 2H),5.74-5.87 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 0.0, 16.4, 25.8, 28.4,28.7, 28.8, 29.2, 29.4, 33.6, 69.4, 69.8, 70.0, 70.4, 71.3, 84.4, 106.6,113.9, 138.9. ESI-MS: m/z 758 [M+Na]⁺.

At −78° C., n-butyllithium (2.2 M in hexane, 1.1 mL, 2.4 mmol) was addedto a solution of S12 (200 mg, 0.3 mmol) in dry THF (20 mL) with 3 Å MS(20 mg). After stirring for 1 h at this temperature,chlorodimethyl-3,3,3-trifluoropropylsilane (419 mg, 2.2 mmol) was added.The reaction mixture was warmed to room temperature and stirred overnight. Usual workup and flash chromatography (ethylacetate/methanol=100/3) afforded 4 (50 mg, 20% yield) as colorless oil.¹H NMR (300 MHz, CDCl₃) δ 0.15 (s, 6H), 0.75-0.81 (m, 2H), 1.23-1.37 (m,10H), 1.51-1.58 (m, 2H), 1.73-1.82 (m, 2H), 1.98-2.14 (m, 4H), 2.31 (t,J=7.2 Hz, 2H), 3.43 (t, J=6.6 Hz, 2H), 3.50-3.69 (m, 42H), 4.89-5.00 (m,2H), 5.72-5.86 (m, 1H). ESI-MS: m/z 841 [M+Na]⁺.

At −76° C., to a stirred soln. of S12 (51 mg, 0.077 mmol) in dry THF (1ml) under nitrogen was added a soln. of LDA (1.8 M inTHF/benzene/haptane, 0.12 ml, 0.216 mmol). After stirring for 2 h at−76° C., Me₃GeCl (32 μl, 0.259 mmol) was added. The mixture was stirredat −76° C. for 2 h, and allowed to warm up to room temperature andstirred overnight. Usual workup and flash chromatography (methanol/ethylacetate=1/99) afforded 5 (22 mg, 37% yield) as pale yellow oil. ¹H-NMR(300 MHz, CDCl₃) δ 0.30 (s, 9H), 1.24-1.37 (m, 12H), 1.53-1.58 (m, 2H),1.72-1.80 (m, 2H), 1.98-2.05 (m, 2H), 2.29 (t, J=6.9 Hz, 2H), 3.43 (t,J=6.6 Hz, 2H), 3.50-3.64 (m, 40H), 4.89-5.01 (m, 2H), 5.72-5.86 (m, 1H).¹³C-NMR (300 MHz, CDCl₃) δ 26.06, 28.76, 28.89, 29.04, 29.42, 29.61,33.78, 69.79, 70.02, 7.17, 70.54-70.59 (m), 71.51, 84.29, 105.24,114.10, 139.18. MS (ESI): m/z 798 [M+H₂O]⁺.

To a solution of S13⁴ (100 mg, 0.37 mmol) in dry THF (1.0 mL) at roomtemperature was added trifluoroacetic anhydride (116 mg, 0.56 mmol). Thereaction mixture was stirred overnight, concentrated in vacuum. Theresidue was purified by silica gel column chromatography to give 6 (141mg, 95%) as a colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ 7.26-7.40 (m,1H), 3.69-3.56 (m, 20H), 3.53-3.50 (m, 2H), 3.34 (m, 2H); ¹³C NMR (75MHz, CDCl₃) δ 157.3 (q, J=37.5 Hz), 115.9 (d, J=287 Hz), 71.2, 70.6,70.5, 70.5, 70.4, 70.3, 70.2, 69.9, 68.8, 50.6, 39.7. ESI-MS: m/z 425[M+Na]⁺.

Powdered molecular sieves (3 Å) and S2 (308 mg, 0.875 mmol) were addedto a stirred solution of S14 (240 mg, 0.584 mmol) in dry CH₂Cl₂ (5 mL).After 15 min, HgBr₂ (210 mg, 0.584 mmol) was added. The mixture wasstirred overnight, diluted with CH₂Cl₂ (20 mL) and filtered over a padof celite. The organic phase was washed with 5% KI (3×15 mL) and water(3×15 mL), dried over Na₂SO₄. Flash chromatography (EtOAc:CH₂Cl₂=1:1)provided S15 (150 mg, 40%) as light yellow oil. ¹H NMR (300 MHz, CDCl₃)δ 2.00 (s, 3H), 2.05 (s, 3H), 2.11 (s, 3H), 2.16 (s, 3H), 3.46 (t, J=4.8Hz, 2H), 3.68-3.73 (m, 26H), 4.06-4.12 (m, 2H), 4.30 (dd, J=6.0, 12.9Hz, 1H), 4.88 (s, 1H), 5.26-5.33 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ20.58, 20.63, 20.7, 20.8, 50.6, 62.5, 63.4, 66.2, 67.0, 68.6, 69.1,69.2, 69.5, 69.66, 69.71, 69.73, 69.8, 69.9, 70.0, 97.5, 169.6, 169.9,170.0, 170.6. ESI-MS: m/z 638 [M+1]⁺. ESI-HRMS: Calcd. for C₂₆H₄₄O₁₅N₃:638.2774. found: m/z 638.2645.

To a solution of S15 (110 mg, 0.18 mmol) in MeOH (4 mL) was added MeONa(49 mg, 0.72 mmol) at 0° C. The mixture was allowed to warm to roomtemperature and stirred overnight. The mixture was diluted with ether,and neutralized with HCl. Flash chromatography (EtOAc:MeOH=20:1 to 15:1)provided 7 (40 mg, 48% yield) as colorless oil. ¹H NMR (300 MHz, CDCl₃)δ 3.41 (t, J=5.4 Hz, 2H), 3.58-3.71 (m, 24H), 3.89 (s, 2H), 4.00 (s,2H), 4.89 (s, 1H), 6.27 (s, 5H). ¹³C NMR (75 MHz, CDCl₃) δ 50.6, 60.0,65.9, 66.7, 69.8, 70.1, 70.2, 70.35, 70.44, 72.4, 100.0. ESI-MS: m/z 492[M+Na]⁺.

General Procedure for Surface Hydrosilylation to Deposit TMG-TerminatedMonolayer on Hydrogen-Terminated Silicon (111) Surfaces

Briefly, a commercial silicon (111) wafer (single side polished Si,thickness of 500-550 μm, boron-doped (p-type), 1-10 Ω-cm resistivity,miscut angle ±0.5°) was cut into pieces of 2×2 cm², and cleaned withPiranha solution (concentrated H₂SO₄ and 30% H₂O₂ 3:1 (v/v)) at 80° C.for 20-30 minutes to remove organic contaminates. (Caution: sincePiranha solution reacts violently with many organic compounds, extremecare must be taken when handling it). The freshly cleaned sample wasimmersed in an Argon-saturated, 40% NH₄F solution for 15 min followed byrapid rinse with Argon-saturated Millipore water and dried with a streamof nitrogen. This H—Si substrate was immediately transferred into ourhome-built vacuum chamber. After degassed for 10 mM at 10′ Torr, thesubstrate was brought in contact with a droplet (ca. 2-3 mg) of alkenes2, 3, 4, or 5 on a quartz window, forming a uniform layer of the alkenesandwiched by the quartz window and the silicon substrate.Hydrosilylation was performed under 254 nm UV illumination for 2 h. Thesample was washed thoroughly with dichloromethane and absolute alcoholfollowed by drying under a stream of argon.

Surface Click Reaction.

In a typical procedure, to a 4 ml vial containing the TMG-terminated Si(111) substrate as prepared above were added 200 μL of Cu(MeCN)₄ PF₆ (5mM), the Cu(I) ligand 28 (25 mM), and ascorbic acid (50 mM) in degassedmethanol. Note: The methanol was freeze-thaw pumped for at least 6×prior to mixing. After incubation for 10 minutes, 250 μL of theCF3-terminated azide 6 (10 mM) in ethanol/water (1:1 v/v) was added.After incubation for 4 h under inert (N₂) atmosphere, 200 μL of 5 mMN,N,N′,N′-tetramethylethane-1,2-diamine (EDTA) was added. The sample wastaken out and washed by sonication for 10 seconds in a mixture ofethanol and methanol (˜1:1 v/v) and then Millipore water. Finally, thefilms were dried under a stream of argon.

X-Ray Photoelectron Spectroscopy (XPS).

A PHI 5700 X-ray photoelectron spectrometer, equipped with amonochromatic AlKα X-ray source (hv=1486.7 eV) at a take-off angle (TOA)of 45° from the film surface, was employed for XPS measurement.High-resolution XPS spectra were obtained by applying a window passenergy of 23.5 eV and the following numbers of scans: Si2p, 3 scans;C1s, 6 scans; O1s, 3 scans; N 1s, 30 scans; Ge3d, 15 scans; F1 s, 6scans. The binding energy scales were referenced to the Si2p peak at99.0 eV. XPS spectra were curve fitted and the intensities measured aspeak areas were calculated using Phi Multipak V5.0A from PhysicalElectronics.

Ellipsometry.

An ellipsometer (Rudolph Research, Auto EL III), operated with a 632.8nm He—Ne laser at an incident angle of 70°, was employed for thicknessmeasurement. The refractive index of the silicon films was 3.839. Atleast there measurements were taken for each sample, and the mean valueswere reproducible within ±1 Å.

Atomic Force Microscopy.

The films obtained before and after click reaction were examined using aMultiMode (Digital Instruments Inc., Santa Barbara, Calif., USA) atomicforce microscope with a Nanoscope Ma (Digital Instruments Inc)controller and a scanner with a maximum xy scan range of 17×17 μm.Images were acquired in tapping mode using a silicon nitride cantilever(MikroMasch, San Jose, Calif., USA) with a resonance frequency of 132.9KHz and a nominal force constant of 1.75 N/m. Depending on the imagescanned, the scan area varied from 250 nm²-10 μm² and the scanning ratefrom 1.97-1.49 Hz. Initially, the samples were scanned in a large area(10 μm×10 μm) at several locations to verify the homogeneity of thesurface. Then, high resolution scans were performed at slower scan rate(1.49 Hz) to obtain representative images of the acquired films.

Fluorescence Microscopy.

Fluorescence images were obtained with an Olympus BX 51 fluorescencemicroscope with a 60× objective. Filter FITC was used for exciting GFP.The exposure time for the acquisition of each image was 0.01-1 s. Imageswere processed using QCapture software.

It will be understood that certain of the above-described structures,functions, and operations of the above-described embodiments are notnecessary to practice the present invention and are included in thedescription simply for completeness of an exemplary embodiment orembodiments. In addition, it will be understood that specificstructures, functions, and operations set forth in the above-describedreferenced patents and publications can be practiced in conjunction withthe present invention, but they are not essential to its practice. It istherefore to be understood that the invention may be practiced otherwisethan as specifically described without actually departing from thespirit and scope of the present invention as defined by the appendedclaims.

What is claimed is:
 1. A catalyst effective for a 1,3-dipolarcycloaddition reaction, wherein the catalyst comprises Cu(I) and atriazole ligand comprising oligoethylene glycol, wherein the triazoleligand is selected from


2. The catalyst according to claim 1, wherein the 1,3-dipolarcycloaddition reaction comprises functionalizing regions of an OEGmonolayer with a molecular probe, wherein the molecular probe serves asrecognition element for a bioanalyte.
 3. The catalyst according to claim2, wherein the regions are distal to a surface, wherein a silicon (Si)substrate comprises the surface.
 4. The catalyst according to claim 1,wherein the trizole ligand is water soluble.
 5. A catalyst effective fora 1,3-dipolar cycloaddition reaction, wherein the catalyst comprisesCu(I) and a ligand selected from among

wherein the 1,3-dipolar cycloaddition reaction comprises functionalizingregions of an OEG monolayer with a molecular probe, wherein themolecular probe serves as recognition element for a bioanalyte, whereinthe regions are distal to a surface, wherein a silicon (Si) substratecomprises the surface.